Biosynthesis of a rare di-N-acetylated sugar in the lipopolysaccharides of both Pseudomonas aeruginosa and Bordetella pertussis occurs via an identical scheme despite different gene clusters

Abstract

Pseudomonas aeruginosa and Bordetella pertussis produce lipopolysaccharide (LPS) that contains 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid (D-ManNAc3NAcA). A five-enzyme biosynthetic pathway that requires WbpA, WbpB, WbpE, WbpD, and WbpI has been proposed for the production of this sugar in P. aeruginosa, based on analysis of genes present in the B-band LPS biosynthesis cluster. In the analogous B. pertussis cluster, homologs of wbpB to wbpI were present, but a putative dehydrogenase gene was missing; therefore, the biosynthetic mechanism for UDP-D-ManNAc3NAcA was unclear. Nonpolar knockout mutants of each P. aeruginosa gene were constructed. Complementation analysis of the mutants demonstrated that B-band LPS production was restored to P. aeruginosa knockout mutants when the relevant B. pertussis genes were supplied in trans. Thus, the genes that encode the putative oxidase, transaminase, N-acetyltransferase, and epimerase enzymes in B. pertussis are functional homologs of those in P. aeruginosa. Two candidate dehydrogenase genes were located by searching the B. pertussis genome; these have 80% identity to P. aeruginosa wbpO (serotype O6) and 32% identity to wbpA (serotype O5). These genes, wbpO(1629) and wbpO(3150), were shown to complement a wbpA knockout of P. aeruginosa. Capillary electrophoresis was used to characterize the enzymatic activities of purified WbpO(1629) and WbpO(3150), and mass spectrometry analysis confirmed that the two enzymes are dehydrogenases capable of converting UDP-D-GlcNAc, UDP-D-GalNAc, to a lesser extent, and UDP-D-Glc, to a much lesser extent. Together, these results suggest that B. pertussis produces UDP-D-ManNAc3NAcA through the same pathway proposed for P. aeruginosa, despite differences in the genomic context of the genes involved.

FIG. 1.

Proposed biosynthetic pathway for UDP-d-ManNAc3NAcA in P. aeruginosa serogroup O2 and B. pertussis. Protein names are indicated in bold for P. aeruginosa and in normal text for B. pertussis. WbpA is underlined to show that it lacks a B. pertussis homolog within the band A trisaccharide biosynthesis cluster.

FIG. 2.

Comparison of LPS biosynthetic loci from P. aeruginosa and B. pertussis (32, 42). (A) B-band O antigen gene cluster from P. aeruginosa PAO1 (serotype O5). (B) Band A trisaccharide gene cluster from B. pertussis Tohama I, also known as the wlb locus. Initial proposed functions for the genes in these clusters were provided from sequence and mutational analysis of P. aeruginosa and B. pertussis, respectively (4, 35). Genes known or predicted to be involved in the biosynthesis of UDP-d-ManNAc3NAcA are shaded gray; genes known or predicted to encode dehydrogenases are shaded gray with black hatching.

FIG. 3.

Cross-complementation of wbpA knockouts in P. aeruginosa PAO1 with plasmid-encoded His₆-WbpA or either His₆-WbpO₁₆₂₉ or His₆-WbpO₃₁₅₀ from B. pertussis. (A) Silver-stained SDS-PAGE gel; (B) Western blot with anti-B-band O antigen MAbs. B-band O antigen was not detected in the wbpA knockout strain or the knockout transformed with empty vector; B-band O antigen was detected when the knockout strains were complemented with plasmid-encoded His₆-WbpA, His₆-WbpO₁₆₂₉, or His₆-WbpO₃₁₅₀.

FIG. 4.

Cross-complementation of various wbp knockouts in P. aeruginosa PAO1 by homologs from the wlb locus of B. pertussis. (A) Silver-stained SDS-PAGE gel; (B) Western blot with anti-B-band O antigen MAbs. B-band O antigen was not detected in the knockout strains or knockout strains transformed with an empty vector; B-band O antigen was detected when the knockout strains were complemented with the relevant B. pertussis gene.

FIG. 5.

SDS-PAGE analysis of purified dehydrogenase proteins after Ni⁺-affinity chromatography and buffer exchange according to the work of Miller et al. (31). Proteins were expressed with a hexahistidine tag from pET-28a. All proteins were free of contamination.

FIG. 6.

Electropherograms from CE of dehydrogenase reactions. (A) Control reaction mix (no enzyme) with NAD⁺ and UDP-d-Glc. (B) Reaction with His₆-WbpO₃₁₅₀, NAD⁺, and UDP-d-Glc yielded UDP-d-GlcA. (C) Control reaction mix with NAD⁺ and UDP-d-GlcNAc. (D) Reaction with His₆-WbpO₃₁₅₀, NAD⁺, and UDP-d-GlcNAc yielded UDP-d-GlcNAcA. The same reaction is catalyzed by His₆-WbpA, as previously shown by Miller et al. (31). (E) Control reaction mix with NAD⁺ and UDP-d-GalNAc. (F) Reaction with His₆-WbpO₃₁₅₀, NAD⁺, and UDP-d-GalNAc yielded UDP-d-GalNAcA. The same reaction is catalyzed by P. aeruginosa His₆-WbpO, as previously shown by Miller et al. (30).